The Specific Protein Kinase R (PKR) Inhibitor C16 Protects Neonatal Hypoxia-Ischemia Brain Damages by Inhibiting Neuroinflammation in a Neonatal Rat Model

Abstract

BACKGROUND Brain injuries induced by hypoxia-ischemia in neonates contribute to increased mortality and lifelong neurological dysfunction. The specific PKR inhibitor C16 has been previously demonstrated to exert a neuroprotective role in adult brain injuries. However, there is no recent study available concerning its protective role in hypoxia-ischemia-induced immature brain damage. Therefore, we investigated whether C16 protects against neonatal hypoxia-ischemia injuries in a neonatal rat model. MATERIAL AND METHODS Postnatal day 7 (P7) rats were used to establish classical hypoxia-ischemia animal models, and C16 postconditioning with 100 ug/kg was performed immediately after hypoxia. Western blot analysis was performed to quantify the phosphorylation of the PKR at 0 h, 3 h, 6 h, 12 h, 24 h, and phosphorylation of NF-κB 24h after hypoxia exposure. The TTC stain for infarction area and TUNEL stain for apoptotic cells were assayed 24 h after the brain hypoxia. Gene expression of IL-1β, IL-6, and TNF-α was performed at 3 h, 6 h, 12 h, and 24 h. RESULTS The level of PKR autophosphorylation was increased dramatically, especially at 3 h (C16 group vs. HI group, P<0.01). Intraperitoneal C16 administration reduced the infarct volume and apoptosis ratio after this insult (C16 group vs. HI group<0.01), and C16 reduced proinflammatory cytokines mRNA expression, partly through inhibiting NF-κB activation (C16 group vs. HI group<0.05). CONCLUSIONS C16 can protect immature rats against hypoxia-ischemia-induced brain damage by modulating neuroinflammation.

Figure 1

The flow chart of the study.

Figure 2

The level of PKR autophosphorylation in neonatal brain I induced by hypoxia-ischemia. The level of PKR autophosphorylation were detected at different time points (0 h, 3 h, 6 h, 12 h, and 24 h) after model establishment (A) Representative images of phosphorylated and total forms of PKR. (B) Ratio of pPKR/PKR calculated from the densitometric analyses of blots. * P<0.05 vs. Control group, ** P<0.01 vs. Control group.

Figure 3

The effects of C16 administration on brain tissue loss at 24 h after hypoxia-ischemia. (A) Representative samples of TTC-stained coronal sections were obtained 24 h after hypoxia-ischemia. Notable cerebral infarction region was observed in the HI group. (B) After HI, the infarct ratio was increased dramatically, and the infarct ratio was clearly lower after intraperitoneal C16 administration. ** P<0.01 vs. Control group, ^## P<0.01 vs. HI group.

Figure 4

The effects of C16 postconditioning on apoptosis of brain tissues at 24 h after hypoxia-ischemia. (A) Representative samples of TUNEL-stained coronal sections. TUNEL-positive cells showed niger-brown staining (arrow). (B) The IOD/area in the HI group was significantly elevated, while the C16 group showed a dramatic downregulation. ** P<0.01 vs. Control group, ^## P<0.01 vs. HI group.

Figure 5

C16 administration modulated the cytokines mRNA expression. Cytokines mRNA were detected at different time points (0 h, 3 h, 6 h, 12 h, and 24 h) after model establishment. (A) TNF-α, (B) IL-1β, (C) IL-6. Inflammatory cytokines mRNA expression levels increased gradually from 0 h to 24 h, while C16 postconditioning restrained the cytokines mRNA expression. * P<0.05 vs. C16 group. ** P<0.01 vs. C16 group.

Figure 6

The effects of C16 on NF-κB activity at 24 h after hypoxia-ischemia. (A) Representative images of pP65 and P65. (B) The phosphorylation levels of P65 were increased in the HI group, while in the C16 group the expression levels of pP65 were restrained. * P<0.05 vs. Control group, ^# P<0.05 vs. HI group.